1. Field of the Invention
The invention is directed to the systems, components and methods for in vitro culturing of eukaryotic cells and, in particular, mammalian cells for the production of bulk quantities of virus. In addition, these systems, components and methods are useful for the propagation of virus from cell cultures by elimination of cellular DNA as well as residual proteins to obtain and isolate virus and viral components, preferably in an inactivated form, for the manufacture of vaccines.
2. Description of the Background
The ability to grow viruses and culture cells in vitro has been one of the single greatest advances in biology and medicine. Cell culturing has led to an understanding of the functions of cellular mechanisms, unraveling the processes cell-to-cell interactions, drug discovery and even the ability to develop and manufacture vaccines.
Currently, there are a great many variations in the in vitro culture of eukaryotic cells. Most advances in cell culture today are directed to addressing the specific requirements of one cell or tissue type. These advances include the use of incubators, capillaries, and micro-carriers, variations in culture media, cell-adhesion materials such as matrixes, the use of a movable bag referred to as a wave bioreactor and others (e.g., BioWave, Wave Bioreactor, BIOSTAT CultiBag, AppliFlex, Cell-Tainer, Tsnumai-Bioreactor, Optima, and Orbicell,). Each of these techniques addresses a unique problem encountered with a particular cell or tissue type. However, the basic concerns in achieving a successful cell culture after virus inoculation remains largely the same.
With all virus infected cells grown in vitro, there are at least six principle areas to be addressed: (i) appropriate starter cells are necessary; (ii) the critical mass of the cell culture needs to be determined; (iii) cells must be readily supplied with nutrients including necessary vitamins and minerals as well as oxygen; (iv) cell waste products must be efficiently removed; (v) stress on the cell cultures attributable to the incubation system should be minimized or eliminated, such as shear forces and hydrostatic pressures, (vi) sterility must be maintained throughout which includes complete separation from not only contamination in the form of bacteria and virus, but isolation from other cells and cell cultures. The infection of these cells with a virus for the purpose of manufacturing a vaccine adds the additional steps of inactivating the virus, the elimination of cellular DNA as well as residual proteins to obtain and isolate virus and viral components for a usable therapeutic vaccine and ensuring that the system operates in a safe manner for those working with potentially hazardous viruses.
The choice of appropriate cells for culturing and virus infection has remained a significant issue for decades. Currently, most virus infected cells obtained from a biological sample are maintained, if only for short periods of time. Many tissue types are harvested after growth and virus inoculation with the goal of seeking viable cell cultures. As yet there are no well-defined markers for determining viability in virus inoculated cell culture.
Supplying nutrients to the cells and removing cell waste products efficiently has been the subject of a great deal of research with much success. The issue of supplying nutrients such as carbohydrates, lipids, minerals, and vitamins has been successfully resolved by several techniques, such as, for example, hollow fiber technology. These and other advantages have been successfully incorporated into many cell culturing systems and techniques. However, the principles determining maximum cell mass after virus infection is closely related to the ability of the system to provide readily available nutrients including oxygen, and also remove waste products.
In the virus infected cell culture systems, stress on the cells should be minimized or eliminated. The presence of stresses, such as shear forces and hydrostatic pressures, greatly affects the efficiency of the culture system, and the amount of final virus yields derived which can increase from each run. Prior methods of reducing shear and hydrostatic forces include developing new oxygenation devices in order to reduce shear caused by bubbles, exploiting different protective agents, and modifying existing impellers and designing new types of agitators and immobilizing cells within a carrier matrix.
Conventional cell culture techniques typically involve a situation of no stress or stress. In the no stress situation, cells grow in relative stasis and without stress damage, but due to inadequate media exchange are less vigorous and proliferate poorly. In the stress situation, although the media exchange and removal of cell metabolites is good for vigorous cell growth, shear forces disengage the cells from anchorage surfaces and these detached cells perish which diminishes the amount of final viral yield.
The implementation of facile sterilization procedures for bioreactors and associated components is essential for clinical utility of inoculated virus cells to be used as a vaccine. The procedures for sterilization are well established, including standard methods both for sterilization of extracorporeal devices and for maintaining asepsis by standard in-line filters.
The inactivation of the cultured viral product is often achieved by solvent/detergent inactivation, pasteurization, pH activation or ultraviolet inactivation and chemical treatment. The inactivation method chosen often depends on the desired viral product. The solvent/detergent method is effective with viruses containing a lipid envelope, as the detergents used interrupt interactions between the molecules in the virus's lipid coating. As most enveloped viruses cannot live without their lipid coating, they die when exposed to these detergents. Common detergents used in this method include Triton-X 100. Pasteurization can also be effective if the desired proteins are ore thermally resistant than the viral impurities with which they are in solution. Certain proteins act as thermal stabilizers for viruses. Additionally, if the target protein is not heat-resistant, pasteurization could denature the target protein as well as the viral impurity. With pH inactivation, some viruses will denature spontaneously when subjected to low pH (acidic). This technique is useful if the target protein is more resistant to low pH than the viral impurity, and is generally more effective against enveloped viruses as opposed to unenveloped viruses. Ultraviolet inactivation can be used to induce the dimerization of nucleic acids within the virus. Once the DNA is dimerized, the virus particles cannot replicate their genetic material, preventing infection. Among the known chemical inactivation formaldehyde and Beta Propiolactone have been used for very many virus inactivations.
Vaccine Production
Each day, the human body is attacked by bacteria, viruses or other infectious agents. When an individual becomes infected with a disease causing agent, the body's built-in immune system attempts to defend against the foreign agent. When the body successfully defends itself, immunity against the infectious agent results. When the body's natural defenses fail to quell the attack, an infection can be the results. In the natural process of developing immunity, B cells produced by the body produce substances known as antibodies that act against the specific infectious agent and create a “log” of this experience that can be called upon for protection when exposed to the same infectious agent again months, years or even decades later. For subsequent times that the person encounters that specific infectious agent, circulating antibodies quickly recognize the infection and eliminate that infection from the body before signs of disease develop. It has been estimated that antibodies which can recognize as many as 10,000 different antigens or foreign infectious agents are circulating the blood stream.
A vaccine works in a similar manner in that it induces the body to generate an immunogenic response. However, instead of initially suffering the natural infection and risking illness in order to develop this protective immunity, vaccines create a similar protective immunity without generally exposing the body to a condition wherein an infection could occur.
Development of vaccines against both bacterial and viral diseases has been one of the major accomplishments in medicine over the past century. While conventional procedures have allowed for the development of effective vaccines for a number of diseases, these procedures have been ineffective for others. Thus a need remains for the development of safe and effective vaccines for a number of additional diseases.
Several basic strategies are useful in the manufacture of vaccines. One strategy is directed toward preventing viral diseases by weakening or attenuating a virus so that the virus reproduces very poorly once inside the body. Measles (Morbilliviruses virus), mumps (which can be viral or bacterial), rubella (Rubivirus or German measles) and chickenpox (varicella zoster virus) vaccines are made this way. Whereas natural viruses usually cause disease by reproducing themselves many thousands of times, weakened vaccine viruses reproduce themselves approximately 20 times. Such a low rate of replication is generally not enough to cause disease. Although the preparation of live, attenuated infectious agents as vaccines will often provide improved immunologic reactivity, such methods increase the risk that the vaccine itself will be the cause of infection, and that the attenuated organism will propagate and provide a reservoir for future infection. One or two doses of live “weakened” or attenuated virus may provide immunity that is life-long; however, such vaccines cannot be given to people with weakened immune systems.
Another method to manufacture viral vaccines is to inactivate the wild-type virus and use the inactivated materials to generate an immune response. By this method, viruses are completely inactivated or killed using a chemical. Killing the virus makes the virus unable to replicate in a body and cause disease. Polio, hepatitis A, influenza and rabies vaccines are made this way. The use of inactivated or killed bacterial or viral agents as a vaccine, although generally safe, will not always be effective if the immunogenic characteristics of the agent are altered. An inactive virus can be given to people with weakened immune systems, but may have to be given multiple times to achieve immunity.
Vaccines may also be made using parts of the virus. With this strategy, a portion of the virus is removed and used as a vaccine. The body is able to recognize the whole virus based on initial exposure to a portion of the virus. The hepatitis B vaccine for example, is composed of a protein that resides on the surface of the hepatitis B virus.
Thus, one must generally choose between an improved effectiveness and a greater degree of safety when selecting between the inactivation and attenuation techniques for vaccine preparation. The choice is particularly difficult when the infectious agent is resistant to inactivation and requires highly rigorous inactivation conditions which are likely to degrade the antigenic characteristics which help to induce an immune response and provide subsequent immunity.
In addition to the dead or weakened infectious agent, vaccines usually contain sterile water or saline. Some vaccines are prepared with a preservative or antibiotic to prevent bacterial growth. Vaccines may also be prepared with stabilizers to help the vaccine maintain its effectiveness during storage. Other components may include an adjuvant which helps stimulate the production of antibodies against the vaccine to make it more effective.
Methods to prepare vaccines today involve treating samples with glutaraldehyde or formaldehyde to fix or cross-link the cells or infectious particles. Such treatments generally involve denaturation of the native forms of the infectious particles. A disadvantage to this approach is that the protein coats of the infectious particles are damaged by this process, and thus may not be recognized by the immune system.
A clear need exists for a complete, modular, scalable system to handle large-scale bulk manufacturing of viruses including bio-safety level 3 viruses safely and efficiently. The problems with existing bioreactor designs include inadequate oxygenation, minimal capacity for the biological cell component, limited capability for removal of toxins, excessive shear and hydrostatic forces, and difficulty in transferring biosynthetic cell products for patient use. In addition, existing bioreactor designs have not dealt effectively with the diffusion-limited thickness of the cell mass, with providing a critical mass of cells, and with supplying oxygen throughout the length of the bioreactor.
Thus, there is a need for a composite apparatus that is able to carry out the entire process of cell growth, virus infection and propagation, purification and inactivation in a closed, low- or no-stress apparatus that maximizes cell growth and virus propagation, and increase throughput, recovery and safety.